We are investigating the evolution of viral genetic diversity in HIV infection and multidrug resistance in patients on therapy. A repository of specimens from Cameroon that have been screened by a rapid HIV test currently in use in blood banks in Cameroon are being characterized by sequencing in the gag, pol and env region in order to identify their subtype and recombinant forms. Cameroon was selected for this study because of the high prevalence of multiple diverse subtypes in this region. The potential for recombinant forms to evolve would be expected to be high in such a setting. Approximately 150 screened positive and 100 screened negative specimens were tested on a number of licensed antibody, antigen and NAT assays. Most specimens were detected by the antibody and NAT assays although some were discordant and are under investigation by sequence analysis. All but one of the specimens were negative on p24 antigen tests. Sequence analysis revealed that most of the 150 positive specimens were recombinant A/G but novel A/C forms were identified. We are culturing virus from the sera for studies on virus biology including cytokine and chemokine profiles of these variant viruses. The prevalence of HTLV and HHV-8 as coinfections with HIV are also under investigation. HIV negative specimens are being evaluated for the presence of novel retroviral agents by a generic PCR assay. We are developing a microarray for detection of the various HIV-1 and -2 subtypes, HTLV and HHV-8 to expedite analysis of these strains. Future efforts will include application of nanotechnology to the detection of these pathogens. In a separate study, multidrug resistant HIV viruses were isolated from a group of patients and their biologic characteristics in regard to co-receptor usage, and replication capacity were studied. Most viruses could be cultured in vitro but although many of them exhibited an NSI phenotype some viruses showed neither X4 nor R5 properties suggesting that they may have evolved new co-receptor usage subsequent to treatment. We are currently exploring the role of other receptors and the potential for these viruses to establish reservoirs in other cell types. We are also studying the cytokine and chemokine profile and apoptotic potential of these viruses in comparison with naive strains in order to understand the pathogenesis of drug resistant HIV. Mass Smallpox vaccination has been considered as a preventive measure to counter-terrorism involving smallpox. This raises concerns about potential viremia in individuals who present as blood donors. Our study involves testing specimens from individual enrolled in 3 IND trials by Taqman and virus culture assays. Virus-cell interactions and host factors in HIV pathogenesis. We have characterized several new strains of the various HIV subtypes ranging from A-G of group M, isolates of group O and HIV-2. We have also obtained several drug resistant strains of HIV. We are sequencing the regulatory genes tat, nef, vpu and vpr to look for any possible relationships between these sequence s and co-receptor usage. Studies are underway to evaluate the replication of non clade B, O viruses and drug resistant viruses in in vitro cell systems with specific emphasis on their cell trpoism, replication kinetics, chemokine and cytokine usage. We are working with CDC to develop strategies in Cameroon (Africa) to identify new variants and recombinants of HIV and related viruses. These viruses are being investigated for their infectivity, drug susceptibility and pathogenesis in appropriate cell culture systems and animal models. We have examined the impact of HIV variation on apoptosis, chemokine and cytokine production in donor PBLs. We will also examine the role of tat and nef in faciltitating fasL mediated apoptosis in PBLs and dendritic cells. These studies will be also carried out in SCID mice. Effects of tat and nef on the regulation of chemokines in susceptible dendritic cells are being investigated. The infectivity of different HIV subtypes and variants for dendritic cells and macrophages are being studied. Detection, quantitation and characterization of HIV. 1. In-house serologic and genetic assays and Taqman probes are being developed for HIV-1 group O and non clade B subtypes. An in-house HIV group O ELISA and primers and probes for HIV group O and M has been developed. The probes will be used to generate microarray based assays for viral genotype. Oligo Chips are being developed to detect all known HIV group M subtypes, group O subtypes. In addition probes that are subtype specific are also under development. These assays will serve as tools in molecular epidemiologic studies to determine prevalence of non clade B strains particularly HIV-1 group O in endemic areas. We have recently initiated a study to study molecular evolution of HIV in Camerooon a region which harbors several subtypes. In these studies we will be characterizing various strains from specimens collected in Cameroon in order to identify new strains. WE have begun studies to investigate the diagnostic significance of strain diversity by testing sera using FDA licensed antibody and nucleic acid tests. We will also compare the performance of various direct viral marker assays such as PERT, RT-PCR, and p24 assays for their ability to detect viral variants and HIV in the early phase of infection. 2. Panels are being developed for the quantitation of RNA of HIV-1 subtypes. Virus isolates from each subtype have been sequenced to verify the subtype. These isolates are being quantitated in a collaborative study. An HIV-2 panel is also under development. Diagnostic and pathogeneic investigations of vaccinia virus: Implications for blood safety. We have initiated studies to investigate the period of viremia post-vaccination. Cells and plasma from recent vaccinees enrolled in candidate vaccine trials are being collected at various time points 0, 3, 7, 14, 28 and 56 days. Sensitive Taqman and virus culture assays have been developed. Plasma viremia studies indicate that virus was not detected in the vaccinee sera to date. In order to evaluate whether vaccination can result in interference in currently licensed viral marker tests used to screen donors samples from vaccinees are being tested on a variety of licensed tests. These studies are in progress. This project incorporates FY2002 projects 1Z01BP002001-10, 1Z01BP002003-10, and 1Z01BP002020-01.